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Immunofluorescence staining in HEK-293 cells and neurons demonstrates ER retention of the remaining mutants. (A) Immunofluorescence co-localization of CASPR2 and calreticulin in stable HEK-293 cell lines. Cells were stained with anti-calreticulin and anti-FLAG antibody. (B) FLAG and calreticulin pixel co-localization to estimate the amount of ER retention of each mutant. Data are presented as mean + SEM values calculated from z-stacks from three separate cells. Y and N below the mutant name refer to the potential of the variant to be deleterious based on PolyPhen and SIFT algorithms as presented in Table 2 by Bakkaloglu et al. (8). (C) FLAG staining of rat hippocampal neurons transfected with CASPR2-WT and D1129H mutant. Note the different disposition of the mutant protein with respect to the wild type.
Source publication
Although genetic variations in several genes encoding for synaptic adhesion proteins have been found to be associated with autism spectrum disorders, one of the most consistently replicated genes has been CNTNAP2, encoding for contactin-associated protein-like 2 (CASPR2), a multidomain transmembrane protein of the neurexin superfamily. Using immuno...
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Context 1
... * strongly co-localizes with calreticulin before secretion and because it is no longer membrane tethered, it lacks surface staining. Consistent with the Endo-H results, the other CASPR2 variants show variable degrees of ER co-localization with some variants showing co-localization values ranging 45% for T1278I to .70% for both Y716C and R1119H ( Fig. 3A and B). Interestingly, I869T, the variant that, apart from CASPR2-1253 * , is most strongly implicated in disease on the basis of genetic data, shows an intermediate level of ER retention. As such, it seems that different variants may perturb cellular disposition in distinct ways, compounding the enormous complexity underlying ...
Citations
... NRXN1 deletions can be inherited from apparently healthy parents [53]. In addition, incomplete penetrance was observed for variants in CNTNAP2 with presentation in heterozygous carriers ranging from severe to apparently unaffected [70]. ...
Background:
Heterozygous, large-scale deletions at 14q24.3-31.1 affecting the neurexin-3 gene have been associated with neurodevelopmental disorders such as autism. Both "de novo" occurrences and inheritance from a healthy parent suggest incomplete penetrance and expressivity, especially in autism spectrum disorder. NRXN3 encodes neurexin-3, a neuronal cell surface protein involved in cell recognition and adhesion, as well as mediating intracellular signaling. NRXN3 is expressed in two distinct isoforms (alpha and beta) generated by alternative promoters and splicing. MM/Results: Using exome sequencing, we identified a monoallelic frameshift variant c.159_160del (p.Gln54AlafsTer50) in the NRXN3 beta isoform (NM_001272020.2) in a 5-year-old girl with developmental delay, autism spectrum disorder, and behavioral issues. This variant was inherited from her mother, who did not have any medical complaints.
Discussion:
This is the first detailed report of a loss-of-function variant in NRXN3 causing an identical phenotype, as reported for heterozygous large-scale deletions in the same genomic region, thereby confirming NRXN3 as a novel gene for neurodevelopmental disorders with autism.
... However, current knowledge regarding CNTNAP2 protein synthesis and degradation is sparse. A previous study reported that CNTNAP2 was increased by proteasome inhibitor MG132 [47], while the mechanisms underlying CNTNAP2 degradation are not defined. In this study, we systematically investigated the degradation pathway of CNTNAP2 and its CTF in neuronal and nonneuronal cell lines. ...
... Misfolded and mutant proteins, or aged proteins with accumulated damage, are prone to have shorter half-lives because they are selectively targeted to degradation. Some pathogenic CNTNAP2 mutations in ASD have altered half-life and expression levels [47], which could disturb the proteostasis of CNTNAP2 and may contribute to the pathogenesis of ASD. For example, CNTNAP2-D1129H, found in monozygotic twins with ASD, triggered the unfolded protein response and showed enhanced degradation through the proteasome pathway [47]. ...
... Some pathogenic CNTNAP2 mutations in ASD have altered half-life and expression levels [47], which could disturb the proteostasis of CNTNAP2 and may contribute to the pathogenesis of ASD. For example, CNTNAP2-D1129H, found in monozygotic twins with ASD, triggered the unfolded protein response and showed enhanced degradation through the proteasome pathway [47]. Several other pathogenic CNTNAP2 mutations, Y716C, G731S, and R1119H, showed increased imCNT/mCNT ratio compared with the wild-type CNTNAP2 [30]. ...
Contactin-associated protein-like 2 (CNTNAP2) gene, located on chromosome 7q35, is one of the largest genes in the human genome. CNTNAP2 protein is a type-I transmembrane protein specifically expressed in the nervous system, with versatile roles in the axonal organization, synaptic functions, neuronal migration, and functional connectivity. CNTNAP2 has been widely investigated as a risk gene for autism spectrum disorder (ASD), and recent studies also implicated CNTNAP2 in Alzheimer's disease (AD). Knowledge of the regulations on CNTNAP2's life cycle is necessary for understanding the related physiological functions and pathological conditions. However, the mechanisms underlying CNTNAP2 protein degradation remain elusive. Therefore, we systematically investigated the half-life and degradation pathway of the human CNTNAP2 protein. We discovered that CNTNAP2 has C-terminal fragments (CTF), which may have essential physiological functions. Our results demonstrated that CNTNAP2 full-length protein and CTF have a short half-life of about 3-4 h. CNTNAP2 proteins are degraded by the ubiquitin-proteasome system and the macroautophagy-lysosome pathway, while the lysosome pathway is more common for CNTNAP2 degradation. This study will provide novel insights and valuable tools for CNTNAP2 functional research in physiological and pathological scenarios.
... ER stress is a major contributor to ISR activation in ASD. Several genetic models of ASD have been shown to induce ER stress [73][74][75][76], however because ASD is an extremely heterogeneous disorder, any single copy number variant or genetic mutation is found in only a small fraction of ASD cases. Using a multivariate model, a recent study showed that ASD status was able to predict mRNA levels of ER stress genes including PKR-like ER kinase (PERK), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), X-box binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and inositol-requiring enzyme 1 (IRE1). ...
Neurological disorders encompass an extremely broad range of conditions, including those that present early in development and those that progress slowly or manifest with advanced age. Although these disorders have distinct underlying etiologies, the activation of shared pathways, e.g., integrated stress response (ISR) and the development of shared phenotypes (sleep deficits) may offer clues toward understanding some of the mechanistic underpinnings of neurologic dysfunction. While it is incontrovertibly complex, the relationship between sleep and persistent stress in the brain has broad implications in understanding neurological disorders from development to degeneration. The convergent nature of the ISR could be a common thread linking genetically distinct neurological disorders through the dysregulation of a core cellular homeostasis pathway.
... Upon UPR induction, ATF6 is translocated to the Golgi apparatus for further processing by Site-1 and Site-2 proteases (S1P and S2P, respectively), leading to the release of amino-terminal fragment ATF6f with transcription factor functions. The expression of a mutant CASPR2 associated with ASD was shown to increase ATF6 (Falivelli et al., 2012;Canali et al., 2018). The precise role of ATF6 signaling in neuropsychiatric disorders warrants further investigations. ...
... is associated with ASD (Ulbrich et al., 2016;Canali et al., 2018). Induction of the UPR or its signaling cascades have been linked to pathologies associated with neuropsychiatric and cognitive deficits like memory consolidation defects; ASD; schizophrenia; post-traumatic stress disorders; stressinduced mental disorders; bipolar disorder; and impaired social behavior (Grunebaum et al., 2009;Falivelli et al., 2012;Di Prisco et al., 2014;Ulbrich et al., 2016;Wen et al., 2016;Crider et al., 2017;Dong et al., 2018;Shen et al., 2019). ...
... The mutant protein is retained in the ER after synthesis and initiates the UPR. ATF6 signaling was increased in cells transiently expressing mutant CASPR2 (Falivelli et al., 2012). Further studies are warranted to decipher the involvement of ATF6-mediated UPR signaling in synaptic defects and associated neuropsychiatric disorders. ...
Neurons are polarized in structure with a cytoplasmic compartment extending into dendrites and a long axon that terminates at the synapse. The high level of compartmentalization imposes specific challenges for protein quality control in neurons making them vulnerable to disturbances that may lead to neurological dysfunctions including neuropsychiatric diseases. Synapse and dendrites undergo structural modulations regulated by neuronal activity involve key proteins requiring strict control of their turnover rates and degradation pathways. Recent advances in the study of the unfolded protein response (UPR) and autophagy processes have brought novel insights into the specific roles of these processes in neuronal physiology and synaptic signaling. In this review, we highlight recent data and concepts about UPR and autophagy in neuropsychiatric disorders and synaptic plasticity including a brief outline of possible therapeutic approaches to influence UPR and autophagy signaling in these diseases.
... Moreover, the regulation of protein synthesis mediated by the PERK-eIF2α branch was found to be implicated in synaptic plasticity (Di Prisco et al., 2014), suggesting that UPR activation could be involved in the regulation of neuronal functions. Furthermore, ER retention and UPR activation have been observed in HEK293 cells expressing D1129H CASPR2, a variant of the neuronal cell-adhesion protein, contactin-associated protein-like 2 (CASPR2), a member of the NRXN family, highly-linked to ASD (Falivelli et al., 2012). Here we summarize the effects of the mutations found in the NLGNs (Table 1), with respect to protein expression and trafficking, since absence of NLGNs at the cell surface leads to a loss-of-function phenotype, due to the lack of interaction with the synaptic partners. ...
The genetics underlying autism spectrum disorder (ASD) is complex and heterogeneous, and de novo variants are found in genes converging in functional biological processes. Neuronal communication, including trans-synaptic signaling involving two families of cell-adhesion proteins, the presynaptic neurexins and the postsynaptic neu-roligins, is one of the most recurrently affected pathways in ASD. Given the role of these proteins in determining synaptic function, abnormal synaptic plasticity and failure to establish proper synaptic contacts might represent mechanisms underlying risk of ASD. More than 30 mutations have been found in the neuroligin genes. Most of the resulting residue substitutions map in the extracellular, cholinesterase-like domain of the protein, and impair protein folding and trafficking. Conversely, the stalk and intracellular domains are less affected. Accordingly, several genetic animal models of ASD have been generated, showing behavioral and synaptic alterations. The aim of this review is to discuss the current knowledge on ASD-linked mutations in the neuroligin proteins and their effect on synaptic function, in various brain areas and circuits.
https://authors.elsevier.com/a/1btn8Y3M3TxbD
... Several roles for Caspr2 in neural development and synaptic connection have been uncovered recently, such as regulating neuronal migration 26 , glutamate receptor trafficking 27 , and facilitating the formation of new synaptic spines 28 . Caspr2 mutations in human pedigrees associate with neurologic abnormalities including ASD [29][30][31] , and deletion of the gene that encodes Caspr2 has been shown to lead to neurodevelopmental abnormalities in mice 26 . ...
The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.
... It has been established that threonine 1,292 on CNTNAP2's intracellular domain regulates endocytosis through protein kinase C (Bel, Oguievetskaia, Pitaval, Goutebroze, & Faivre-Sarrailh, 2009) and that proper surface targeting of close family member neurexin-1 requires its PDZ-binding motif (Fairless et al., 2008). Furthermore, specific sites on CNTNAP2's extracellular region are also critical for proper secretory and axonal trafficking (Falivelli et al., 2012;Pinatel et al., 2017). Our immunocytochemistry experiments link PAR3-CNTNAP2 complexes with clathrin-coated endosomes, suggesting that PAR3 may, via PDZ binding, be another component critical for CNTNAP2 internalization from the surface membrane. ...
A decade of genetic studies has established Contactin‐associated protein‐like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2’s molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and Partitioning‐defective 3 (PAR3) – a polarity molecule isolated in a yeast‐two hybrid screen with CNTNAP2’s C‐terminus. We provide evidence that the two proteins interact via PDZ domain‐mediated binding, that CNTNAP2+/PAR3+ complexes are largely associated with clathrin‐coated endocytic vesicles in heterologous cells, and that PAR3 causes an enlargement of CNTNAP2 puncta size. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that PAR3 limits the mobility of CNTNAP2. Finally, over‐expression of PAR3 but not a PAR3 mutant lacking all PDZ domains (PAR3∆PDZall) can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that PAR3 regulates CNTNAP2 spatial localization.
... This isoform (called isoform 2) is present in similar amounts in WT and KO mice, but at a more than tenfold lower amount compared with the full length CASPR2 in WT . With 12 putative N-linked glycosylation sites , CASPR2 has also been found to be highly glycosylated (Canali et al., 2018;Falivelli et al., 2012;Lu et al., 2016;Rubio-Marrero et al., 2016). This can largely affect many important factors such as binding properties, tertiary structure and flexibility . ...
Anti-CASPR2 autoimmune limbic encephalitis is a central nervous system disorder, characterized by the presence of autoantibodies (autoAbs) directed against CASPR2 in the serum and cerebrospinal fluid. Elderly men are mostly affected, with epilepsy being the predominant symptom. CASPR2 is a neuronal cell adhesion molecule, known for its role in gathering Kv1 channels, regulators of neuronal excitability, at the juxtaparanodal region of the node of Ranvier, an essential organization for saltatory conduction of nervous influxes. Increasing sets of data in literature point out a role for CASPR2 in synaptic functions and neuronal activity. This could explain the observed epilepsy, a neurological symptom that finds its origin in disturbed neuronal activity, in patients with anti-CASPR2 autoimmune limbic encephalitis. In this work, I used patients’ autoAbs as a tool to investigate the role of CASPR2 in normally developed cultured neurons which also allowed me to assess the effects of patients’ autoAbs on synaptic functions and reveal possible physiopathological mechanisms underlying the disease. I first assessed the effects of patients’ autoAbs on CASPR2 surface expression and distribution and on Kv1.2 channel expression in mature in vitro hippocampal neurons. I provided evidence that inhibitory neurons are positive for both Kv1.2 channels and surface CASPR2, and that patients’ autoAbs increase Kv1.2 expression and do not induce CASPR2 internalization. Secondly, I analyzed effects of patients’ autoAbs on excitatory and inhibitory synapses in vitro, in immature and mature hippocampal neurons. In immature neurons, dendritic spine densities and AMPA receptor content are increased, while in mature neurons alteration of gephyrin suggests disturbed neuronal transmission after treatment with patients’ autoAbs. My results allow for a better understanding of CASPR2 functions in synaptic processes and unravel possible pathological mechanisms regarding how anti-CASPR2 autoAbs lead to the clinical presentation of patients with anti-CASPR2 autoimmune limbic encephalitis
... The extracellular domain of Caspr2 is composed of different structural subdomains and is likely to present an overall compact architecture, suggesting complex structurefunction relationships (23,24). Studies in HEK-293 transfected cells showed that some variants are misfolded and present severe trafficking abnormalities, being largely retained in the endoplasmic reticulum (ER), while others are well localized at the plasma membrane (25). However, the functional consequences of the misfolded variants have not been further evaluated. ...
... ER retention of these variants was further monitored by quantifying the proportion of protein co-localizing with an ER marker (KDEL) in transfected cells (Supplementary Material, Fig. S2A-C). Consistent with previous observations (25), a large proportion of HA-R1119H and HA-D1129H was significantly retained in the ER as compared to WT Caspr2 (Fig. 3B), the retention being higher for HA-D1129H. A third variant, HA-I869T, also appeared largely as an immature form on immunoblotting (Supplementary Material, Fig. S1B and C). ...
... The misfolded ER-retained variant R1119H plays a dominantnegative effect in axon growth, arguing that phenotypes mimicking homozygous CNTNAP2 null mutation may also exist in human. Falliveli et al. previously demonstrated that variants of this category could associate with chaperones in the ER, leading to stimulation of the unfolded protein response and subsequent proteasomal degradation (25). We found that they are able to oligomerize with WT Caspr2 and induce its ER retention, thus leading to a lack or decrease of functional protein at the plasma membrane. ...
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterised by deficits in social communication and repetitive behaviours. Alterations in brain connectivity are the most replicated findings in ASD patients. The CNTNAP2 gene, coding Caspr2, has been associated to ASD, with a large number of missense heterozygous variants identified in patients. However, some missense variants have also been found in the control population, questioning their pathogenicity. Here, using in vitro and in vivo approaches we identify new functions for Caspr2 in axonal development and provided proof of principle that some variants could impact these functions. We report that Caspr2 is involved in axon growth of cortical neurons in culture in a dose-dependent manner, with Cntnap2+/- neurons presenting an intermediate phenotype between wild type and Cntnap2-/- neurons. We also show that Caspr2 is required in vivo for the development of axons projecting into two major interhemispheric myelinated tracts, the corpus callosum and the anterior commissure. Performing morphometric and electron microscopy analyses we detect morphological modifications of these structures and alterations in axo-axonal contacts and axonal diameter in both Cntnap2+/- and Cntnap2-/- mice throughout development. Using in vitro assays and axon growth as read-out, we further show that some of the variants display either a dominant-negative effect or a loss-of-function in a Cntnap2+/- genetic background, suggesting that they could alter brain connectivity and thus contribute to the manifestations of ASD.
... It has been established that threonine 1292 on CNTNAP2's intracellular domain regulates endocytosis through protein kinase C (Bel et al., 2009) and that proper surface targeting of family member neurexin1 requires its PDZ-binding motif (Fairless et al., 2008). Furthermore, specific sites on CNT-NAP2's extracellular region are also critical for proper secretory and axonal trafficking (Pinatel et al., 2017;Falivelli et al., 2012). Our immunocytochemistry experiments link Par3-CNTNAP2 complexes with clathrin-coated endosomes, suggesting that Par3 may, via PDZ binding, be another component critical for CNTNAP2 internalization from the surface membrane. ...
A decade of genetic studies has established Contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2's molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and Partitioning-defective 3 (Par3) - a polarity molecule we isolated in a yeast-two hybrid screen with CNTNAP2's C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+/Par3+ complexes are largely associated with clathrin-coated endocytic vesicles, and that Par3 causes an enlargement of these structures. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that Par3 limits the mobility of CNTNAP2 at endosomes, thus stabilizing it at that location. Finally, expression of Par3 but not Par3DeltaPDZ can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that Par3 regulates CNTNAP2 spatial localization to endocytic compartments.
